An important aspect in heterologous polypeptide production in recombinant microorganism is the selection of the promoter used for controlling the expression of the heterologous nucleic acid sequences which encodes the target polypeptide.
A suitable promoter should be strong. That is, produce the respective mRNA in a high rate allowing production of the polypeptide in high amount. Further, the promoter should be readily to be regulated and start the production of the heterologous polypeptide only upon induction.
However, there is the problem that typically the inducing substrate is a nutrient for the microorganism and is consumed by the microorganism. However, when the medium used for cultivating the microorganism runs out of the inducing substrate, the microorganism deactivates the promoter and, thus, expression of the target polypeptide stops. For avoiding unwanted stop of gene expression, the inducer must be added in high amounts and/or continuously supplemented. The need of high amounts of inducer raises the costs of the fermentation process. Further, use of efficient promoters which, however, require expensive inducers is restricted.
Consequently, for the recombinant polypeptide production host cells are desired wherein the activity of the promoter controlling the expression of the heterologous polypeptide is independent from the presence of the inducer but wherein expression of the heterologous polypeptide can be nevertheless tightly regulated.
WO 2006/133210 A2 relates to a method for producing recombinant peptides in a bacterial host cell utilizing a mannitol, arabitol, glucitol or glycerol-inducible promoter, wherein the bacterial host cell has been rendered incapable of degrading or metabolizing the inducer. According to said method a gene or genes encoding for enzymes required for metabolizing the inducer is genetically altered or deleted in the genome so that the cell cannot express, from its genome, a functional enzyme necessary for metabolizing or degrading the inducer. In order to ensure uptake of the inducer genes related to the transport of the inducer into the cell, are unaffected. This method, however, requires nevertheless addition of inducer for catalyzing activation of the respective promoter controlling expression of the target polypeptide. Further, accumulation of inducer in the cell can negatively affect development of the cell.
Many bacteria are able to utilize different carbon sources. If provided with a mixture of carbon sources the carbon source that allows the most rapid growth (primary carbon source) is selected. Simultaneously the functions involved in the utilization of secondary carbon sources are repressed by a phenomenon called carbon catabolite repression (CCR).
Besides CCR the specific catabolic genes involved in the utilization of a less preferred secondary carbon source are only expressed in the presence of said secondary carbon source. Consequently, expression of genes involved in catabolismn of a secondary carbon source depends on the presence of said secondary carbon source (induction) and the absence of a primary carbon source (catabolite repression).
The publication of Tianqui Sun et al “Characterization of a mannose utilization system in bacillus subtilis”, Journal of Bacteriology, American Society for Microbiology, vol. 192, no 8, Apr. 1, 2010, pages 2128 bis 2139 relates to the identification of the mannose operon and its genes as well as of the promoters PmanP and PmanR regulated by mannose. It is reported that the metabolism of mannose is subject to the phosphoenolpyruvate: carbohydrate phosphotransferase system and that the mannose operon is further subject to carbon catabolite repression. For the characterization and identification of function of the individual genes knockout-mutants were prepared lacking the respective genes and, consequently, lacking the respective proteins encoded by said genes. It was found that deletion of the mannose transporter gene manP resulted in constitutive expression from both the promoters PmanP and PmanR, indicating that the mannose transporter ManP has a negative effect on regulation of the mannose operon and the manR gene encoding for the mannose specific transcriptional regulator ManR.
Tobisch et al. “Regulation of the lic operon of Bacillus subtilis and characterization of potential phosphorylation sites of the LiR regulator protein by site-directed mutangenesis” in Journal of Bacteriology, vol. 181, no. 16, Aug. 19, 1999, pages 4995-5003 reports that exchange of the phosphoryl group binding amino acid in the EIIA domain of the regulator LicR by another amino acid results in activity of the mutant regulator LicR in the absence of inducing substrate.
Görke et al. “Carbon catabolite repression in bacteria: Many ways to make the most out of nutrients” in Nature Reviews. Microbiology, vol. 6, no. 8, August 2008, pages 613-624 relates to Streptococcus mutant strains deficient in mannose transporter EIIAB and the influence on phosphoenolpyruvate: carbohydrate phosphotransferase system. It is shown that the mannose transporter EIIAB is not limited to the phosphorylation of mannose only but likewise phosphorylates glucose, fructose and 2-deoxyglucose. Further, it is shown that even in the absence of the mannose transporter EIIAB mannose can be taken up via the fructose specific transporter EIIFRU.
Deutscher et al “The mechanisms of carbon catabolite repression in bacteria” (Current Opinion in Microbiology, Current Biology LTD, GB, vol. 11, no. 2, Apr. 1, 2008, pages 87-93) gives a general overview of mechanism of carbon catabolite repression in different bacteria, for example E. coli and B. subtilis. 